Dynamic induction of drug resistance through a stress-responsive enhancer in acute myeloid leukemia [RNA-seq]

The drug efflux pump ABCB1 is a key driver of chemoresistance, and high expression predicts for treatment failure in acute myeloid leukemia (AML). We show that both acute and chronic exposure of leukemia cells to daunorubicin activates an integrated stress response-like transcriptional program to induce ABCB1 through remodeling and dynamic activation of an ATF4-bound, stress-responsive enhancer. In primary human AML, stress-responsive ABCB1 enhancers are accessible and acetylated, and exposure of fresh blast cells to daunorubicin induces ABCB1 in a dose-dependent manner. Dynamic induction of ABCB1 by diverse stressors, including chemotherapy, facilitates escape of leukemia cells from targeted third-generation ABCB1 inhibition. Stress-induced up regulation of ABCB1 is mitigated by combined use of pharmacologic inhibitors U0126 and ISRIB, which inhibit stress signaling. TS60 - K562 cells were purchased from DSMZ (Braunschweig, Germany) and cultured in RPMI 1640 medium (Sigma Aldrich) supplemented with 2mM L-Glutamine (Life Technologies) and 10% fetal bovine serum (Sigma Aldrich). Three separate vials of early passage K562 cells were thawed and cultured separately for two weeks. (Figure 1A). Sensitive cell lines were designated K562_S1-3, aliquots were frozen and stored before commencing selection with 10nM daunorubicin (Sigma Aldrich). Whilst under selection, cells were counted and replated twice per week. The daunorubicin concentration was adjusted depending on whether cell numbers were increasing (dose increased by 25-50%), decreasing (dose reduced by 25-50%) or static (dose unchanged). Selection continued until cell lines were capable of expansion in medium containing 500nM daunorubicin. Resistant cell lines were designated K562_R1-3. The time taken to acquire this level of resistance was 106 days (K562_R1 and R3) and 117 days (K562_R2). K562_R1-3 were culture without daunorubicin for 10 days prior to RNA extraction, assessment of daunorubicin IC50 and freezing of aliquots. All cell lines (K562_S1-3, K562_R1-3) were tested for mycoplasma and authenticated by short tandem repeat DNA profiling. Total RNA was extracted from 5x105 K562_S1-3 and K562_R1-3 using QIAshredder spin columns and an RNeasy® Plus Micro kit (Qiagen, Manchester, UK). RNA sequencing was performed using two replicates for K562_R1-3 and a single replicate for K562_S1-3. Prior to sequencing RNA integrity was checked using a 2100 Bioanalyzer (Agilent Technologies – www.genomics.agilent.com). PolyA libraries were prepared using a SureSelect PolyA kit (Agilent Technologies), samples were then barcoded and pooled. Sequencing was performed using a NextSeq desktop sequencing system (Illumina – www.illumina.com). A single run (400M reads) of 75bp paired-end sequencing produced a mean of 45.7M reads per sample.